The long-term goal of this research is to gain a better understanding and control over human embryonic stem cells (hESCs) by identifying and manipulating the key microRNAs required for hESC self-renewal and differentiation. MicroRNAs, small (21-23nt) non-coding RNA molecules that can regulate mRNA translation and stability by binding to the target mRNAs with incomplete complementarity, are required for normal stem cell function in mouse, Drosophila and plants. We have previously shown that microRNAs are required for germline stem cell (GSC) self-renewing divisions in Drosophila. Our preliminary results suggest that microRNAs are also critical for stem cell self-renewing division and differentiation in human ESCs, and the goal of this proposal is to identify the critical microRNAs and their mRNA targets in these cells. Towards this goal, we will analyze the phenotypes observed in hESCs lacking mature microRNAs due to gene knockdown (KD) of microRNA processing enzymes, Dicer or Drosha. We will identify up-regulated mRNAs in these KD-lines by genome wide mRNA profiling and determine whether they are responsible for the Dicer or Drosha phenotypes through loss-of-function (LOF), gain-of-function (GOF) and rescue experiments (Aim1). To identify the critical microRNAs that mediate this regulation we will carry out genome wide QPCR profiling of hESC microRNAs. The function of the hESC enriched microRNAs will be analyzed by LOF, GOF and rescue experiments (Aim2). The two aims will connect: The critical microRNAs should have the capacity to rescue dicer KD phenotypes (Aim 2.2) and may be sufficient to down-regulate the target genes identified in Aim 1.2 (Aim 2.3). This study should result in findings that allow better manipulation and more efficient differentiation of hESC which is of particular importance for regenerative medicine. PUBLIC HEALTH RELEVANCE: This study will be the first analysis of the functions of mature miRNAs in hESCs. Identification of the miRNA function in hESCs holds the future for better control and manipulation of the hESCs in regenerative medicine. This is important due to the troubling fact that embryonic stem cells tend to form tumors when transplanted into adult tissue. microRNAs carry hope as molecules which can control stem cell self-renewing division.